Product details

TumorExoRNA™ Kit (Tumor-derived exosome RNA extraction kit) has been developed for the efficient enrichment of tumor-specific exosome population and RNA extraction from human biofluids and vesicle associated RNAs. Exosome capture occurs on immunobeads pre-coated with exosome associated antibodies enabling for tumor-derived exosome enrichment. The captured exosomes are subsequently lysed with an optimized lysis buffer and total RNA is purified using spin columns with a fast and user-friendly process. Eluted RNA can be used for downstream analyses or stored at -80 °C. Exosome standards for positive control are also included in the kit. All our kits guarantee high specificity for exosomal RNA and high yield of total RNA (including small RNAs) than similar products.

Application Details

Application Details

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Application Notes

Immunocapture and enrichment of tumor-derived exosomes from human biofluids and extraction of vesicle associated RNAs.

Starting from 0.1 mL of biological sample (plasma) per reaction. Whole plasma and serum can be directly used for capture. Concentrated urine samples are recommended prior capture according to our suggested protocol.

Simultaneous miRNA and mRNA profiling (qRT-PCR, RT-PCR, microarray).

Comment

Tumor Exosome immunocapture and RNA extraction, High yield of total RNA (including small RNAs)

Concentrate cell supernatant 10-20 fold in spin concentrator*. *The quantity of exosomes could vary between samples. A larger starting amount of sample should be used if the signal is weak.

Reagent preparation:

Bead Washing Buffer: Dilute 5X to 1X with deionized water. Ensure there is no crystal precipitate. NOTE: If crystals are observed, dissolve them by warming up the concentrated 5X Washing Buffer bottle at 37 °C before proceeding with the dilution. Mix 5 mL of 5x Beads Washing Buffer with 20 mL deionized water for a final volume of 25 ml.

RNA Washing Buffer Solution: Add into the bottle containing RNA Washing Buffer the volume of pure ethanol (96 % ) indicated on the bottle's label to get the final ethanol concentration of approximately 70 %.

Elution Buffer and Lysis Buffer are ready to use.

Exosomes binding:

Purified Exosomes (Lyophilized Standards): Purified exosomes do not require this binding step. If the samples are purified exosomes, skip to RNA extraction directly.

Unfractionated Samples:

Place 0.1 mL up to 1 mL of sample into low-binding tubes (not provided in the kit). Volumes suggested: 0.1 mL up to 0.5 mL for small RNA analysis, 0.5 mL up to 1 mL for mRNA analysis.

Add 1X PBS to the sample to get a final volume of 1 ml. (If you are using 1 mL of plasma, dilution is not necessary).

Spin 5 additional min at 14,000g to eliminate ethanol residues from the column. Discard the flow-throw.

Remove the tube and transfer the spin column into an elution tube.

Elute the column with 15 μL of Elution buffer. Incubate 5 min at room temperature. Spin 2 min at 200g and 1 min at 14,000g. Keep the flow-through. Eluted RNA is now ready for downstream analysis or for storage at -80 °C.

Sensitivity: TotalExoRNATM purified exosome RNA can be quantified and analyzed using NanoDrop spectrophotometer (Thermo Scientific), although the measured concentration values are likely to end toward the bottom limit of detection of the instrument. For better quantification, we recommend the concomitant use of electropherogram-based technologies (eg Bioanalyzer, Agilent Technologies) or fluorimetric technologies (Qubit nano, Thermoscientific). ExoRNATM allows extraction of high quality of exosome-derived RNAs from low volumes of sample and better performance than competitors. Efficiency of TotalExoRNATM kit was tested vs a competitor kit for RNA extraction from healthy donor plasma derived exosomes.

Reagent Preparation

Immunobeads can be stored at 4 °C for up to 8 months.

The RNase free columns and elution tubes must be stored at room temperature.

All the opened buffers and diluted reagents including the bead washing buffer, RNA washing buffer, lysis buffer and the elution buffer should be stored at 4 °C.